Mutations in cell-free DNA - Applications in Cancer

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Hi readers! In today’s post, we will look at mutations that can be detected in cell-free DNA. Mutations are changes in the DNA sequence that can happen due to environmental factors or when DNA is copied. With respect to mutations in cell-free DNA, research has been done primarily in cancer. In cancer, such changes occur in genes that play a role in cell growth and death. During the course of tumor development, a tumor accumulates several mutations. These mutations can be detected in cell-free DNA and this indicates that the cfDNA was released by the tumor.

For example, suppose that we isolated cell-free DNA from a breast cancer patient Z and analyzed it for mutations in the PIK3CA gene. PIK3CA gene plays an important role in mediating pathways responsible for growth and survival of the cell. It is the most commonly mutated gene in breast cancer patients, mutations being detected in about 1/3rd of cases. Now, say that Z has a mutation in PIK3CA gene. Now how can this information help us?



The following are some ways in which cell-free DNA mutations can be used:
  1. Disease diagnosis
  2. Selection of targeted therapies
  3. Serial monitoring of mutations

This infographic shows some ways cell-free DNA can be used in cancer management



Diagnosing cancer

Chances of curing cancer are higher when diagnosed at as early a stage as possible. So, development of new diagnostic tests is based on how early they can detect cancer. New potential biomarkers are evaluated whether they can detect cancer at the early, asymptomatic stages. Similarly, cell-free DNA mutations were also evaluated for this purpose.
For diagnosis, cfDNA samples of persons without known symptoms of disease would be screened for the presence of cancer-associated mutations that could lead to the development of a tumor. Presence of a mutation could indicate the presence of an underlying undetected tumor. 

Modifying the above example, let’s say that patient Z was never diagnosed with cancer and didn’t show any symptoms. Her sample was screened for presence of mutations in genes related to breast cancer. On screening, we detect a PIK3CA mutation in Z’s cell-free DNA, which is known to occur in breast cancer. This indicates that Z may have an undetected breast tumor.

Similar research has been performed in several cancers. Detection of single nucleotide polymorphisms (SNPs) was able to differentiate between breast cancer patients and controls. Other studies have shown that cancer-associated mutations can be detected in cfDNA in early-stage disease, before the presence of symptoms, and up to 2 years before cancer diagnosis. However, use of cell-free DNA for diagnosis is still under research. There are many challenges to be solved and may be some years before cfDNA is used for routine testing.

Selection of targeted therapy

Detection of mutations in cfDNA can help in selecting therapy for the cancer patient. Certain therapies have been developed to target a particular protein which is mutated in cancer but not in normal tissues. These are called targeted therapies. Traditional therapies target cells regardless of whether they are cancer cells or not. Targeted therapies are specific to the particular features of cancer cells. They slow down growth of cancer cells by affecting specific proteins or receptors on/in those cells. This website explains what targeted therapies are and how they work.

So, how does cfDNA help in selecting targeted therapies?  As explained above, cfDNA released from tumor cells can contain cancer-specific mutations. So, doctors would ask for a test to be done, which will check for certain mutations for which targeted therapy is available. If that mutation is detected, then the particular therapy targeting that mutation will be effective.  

Again, using the above example, Z has a PIK3CA mutation. There are some drugs that inhibit the function of mutated PIK3CA protein. So, her doctor may choose to prescribe such PIK3 inhibitors to block the function of the mutated PIK3CA and thereby slow down the growth of the cancer cells.  This is one example of the research under way to test the use of cell-free DNA to help in the selection of targeted therapy.

Serial monitoring of mutations

Because cell-free DNA is obtained in a non-invasive manner, serial blood samples can be obtained to test cell-free DNA during and after treatment. This will help in evaluating whether the patients responded to treatment or not, whether they developed resistance to the treatment, to see whether the disease is progressing into higher stages, presence of minimal residual disease and to detect relapse of cancer.

Treatment monitoring – response and resistance

One main application of cell-free DNA mutations is in monitoring response to treatment. A mutation detected at baseline may not be detected after treatment. This means that the tumor is reducing due to the effect of the treatment. To the contrary, if a patient develops new damaging mutations over the course of treatment, that means the tumor has developed resistance to the treatment. 

As per the above example, suppose that patient Z has been diagnosed with breast cancer. A PIK3CA mutation was detected in her cell-free DNA at baseline. There are two scenarios that can occur here. 

One scenario is that a few days after treatment, the PIK3CA mutation is not detected in her cell-free DNA. This indicates that the tumor cells have been destroyed and hence no DNA fragments (containing the mutated gene sequence) are released into the blood stream. 

Another scenario is that a higher number of DNA fragments containing the mutation are detected after treatment. Or a new damaging mutation in the same/another gene is detected. 

Either way, this indicates that the tumor has developed a new mutation, so that the treatment will not affect it. So, now the tumor has developed resistance to the treatment. This example indicates how cell-free DNA can be used for monitoring the patient’s response to treatment.

In various cancers, researchers studied EGFR mutations in circulating tumor DNA. They found that patients showing partial or complete reduction of the tumor had decreased concentrations of mutated DNA fragments. Increased concentrations indicated that the patients did not respond to treatment. Interestingly, one patient showed an initial response and their treatment was stopped. Soon after, the EGFR mutation re-emerged in this patient, indicating that the tumor had stopped responding to treatment. From these studies, it is evident that monitoring ctDNA during treatment can give us real-time information on treatment response.
Another application is to test a patient before treatment to see if they possess any mutations that may cause drug resistance. Such a screening method will ensure that a patient already having resistance mutations to a particular treatment is given an alternative treatment option. For example, if a mutation causing resistance to treatment A was detected in patient Z, then she can be given treatment B, which will not be affected by the presence of that mutation.

Monitoring disease progression

Monitoring mutations in cell-free DNA can give an indication of disease progression. Imagine that patient Z was prescribed PIK3 inhibitors, and we analyze her cfDNA samples at different time points during her treatment. Suppose we detect a different mutation in PIK3CA or another gene, that was not detected in previous samples. It means that the disease might be progressing. One study found that monitoring the ctDNA mutations during treatment could give early signs of disease progression than radiologic and biochemical tests.

Detection of minimal residual disease and relapse

Monitoring cfDNA mutations can also be used to detect minimal residual disease and even relapse. Sometimes a few cancer cells can develop mechanisms so that they can survive treatment. These cells can persist in the body for a long time, even up to years after treatment. This is called minimal residual disease. At a later point of time, they can grow and result in cancer coming back after the treatment is stopped. This is called relapse or recurrence. Minimal residual disease is a major cause of relapse, particularly in leukemia.

Some studies have found that cfDNA can be useful in detecting minimal residual disease or relapse. One study detected mutations in cfDNA up to 12 years after diagnosis without any evidence of disease. Another study detected PIK3CA variants plasma after surgery, in patients who showed no clinical symptoms. 

So, these are some ways cfDNA can be useful in diagnosing and monitoring cancer. Research is still going on to identify techniques that can effectively detect cfDNA markers. I hope you found this post informative. See you in my next post!


References

  1. Zardavas et al 2014. https://breast-cancer-research.biomedcentral.com/articles/10.1186/bcr3605
  2. Bronkhorst et al 2019. https://www.sciencedirect.com/science/article/pii/S221475351830024X
  3. Aarthy et al, 2015. https://link.springer.com/article/10.1007/s40291-015-0167-y
  4. Yourgenome.org. https://www.yourgenome.org/facts/what-is-a-mutation#:~:text=A%20mutation%20is%20a%20change,UV%20light%20and%20cigarette%20smoke.
  5. Cancercenter.org. https://www.cancercenter.com/treatment-options/precision-medicine/targeted-therapy



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